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1 SCOPE AND FIELD OF APPLICATION

The method specifies the procedure for enumeration of aerobic bacteria, yeast, and mold pre-
sent in cosmetic products.

2 PRINCIPLE
Method for enumeration of microorganisms from cosmetic products is direct colony counts.
Products that are not soluble in water are initially treated to render them miscible before enu-
meration procedures are conducted. The possible inhibition of microbial growth by the sam-
ple shall be neutralized to allow the detection of viable microorganism. In all cases and what-
ever the methodology, the neutralization of the antimicrobial properties of the product shall be
checked and validated. The scheme for these analyses is summarized in Fig 1.

3 MEDIA, REAGENTS, AND APPARATUS

3.1 Media and Reagents :

The following culture media and diluents are suitable for enumeration of aerobic mesophilic bac-
teria, yeast, and mold. Other culture media and diluents may be used if they have been demon-
strated to be suitable for use.

3.1.1 Media for enumeration of bacteria and fungi

3.1.1.1 Malt extract agar (MEA) or other suitable media
3.1.1.2 Potato dextrose agar (PDA) or other suitable media
3.1.1.3 Modified letheen agar (MLA) or other suitable media
3.1.1.4 Sabouraud's dextrose broth or other suitable media
3.1.2 Other media and reagents
3.1.2.1 Aqueous solution of 70% ethanol and 1% HCl (v/v) or 4% iodine in 70%
ethanol solution or 2% glutaraldehyde solution
3.1.2.2 Tween 80 (Polysorbate 80)
3.1.2.3 Ethanol, 95% (v/v)
3.2 Apparatus
3.2.1 Pipets, sterile, 1, 5, and 10 mL, graduated
3.2.2 Gauze pads, sterile, 4 x 4 inch
3.2.3 Gauze pads, sterile, 4 x 4 inch
3.2.4 Sterile instruments: forceps, scissors, scalpel and blades, spatulas, and microspatulas
3.2.5 Test tubes, screw-cap, 16 x 125, and 20 x 150 mm
3.2.6 Dilution bottles, screw-cap
3.2.7 Balance, sensitivity of 0.01 g
3.2.8 Petri dishes, sterile, plastic, 15 x 100 mm
3.2.9 Incubators, 30 ± 2°C and 35 ± 2°C
3.2.10 Laminar flow hood with HEPA filter (if available)

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4 SAMPLE HANDLING

4.1 Analyze samples as soon as possible after receiving them. If necessary, store samples at room
temperature. Do not incubate, refrigerate, or freeze sample before or after analysis.
4.2 Inspect samples carefully before opening and note any irregularities of sample container.

5 PROCEDURE

5.1 General Recommendation

5.1.1 Before opening and removing sample contents, disinfect surface of sample container with
aqueous mixture of 70% ethanol (v/v) and 1% HCl (v/v). Use laminar flow hood if possi-
ble. Leave the cleaned surface to dry before opening.

5.1.2 Weigh 10 g or mL of sample from representative portion of contents for microbial analysis.

5.1.3 For products weighing less than 1 g or mL, analyze entire contents. If only one sample unit
is available and multiple analyses are requested (i.e., microbial, toxicological, and chemi-
cal), take sub-sample for microbiological examination before those for other analyses. In
this situation, amount of sub-sample used for microbial analysis will depend on other analy-
ses to be performed. For example, if total sample content is 5 mL, use 1 or 2 mL portion for
microbial analyses.
5.1.4 The amount of sample and diluent given here can be adjusted according to amount of sam-
ple available. If sample has many sub-samples, amount of test material can be increased and
workload streamlined by compositing. Analysts should use their best judgment as to when
and how much material to composite.

5.2 Sample preparation

5.2.1 Liquids. Decimally dilute 1 mL liquid directly into 9 mL modified letheen broth (MLB) in
20 x 150 mm screw-cap test tube for the 10-1 dilution.
5.2.2 Solids and powders. Aseptically remove and weigh 1 g sample into 20 x 150 mm screw-
cap test tube containing 1 mL sterile Tween 80. Disperse product in Tween 80 with sterile
spatula. Add 8 mL sterile MLB and mix thoroughly. This will be the 10-1 dilution.
5.2.3 Waxy/Fatty products (Lipstick). Aseptically remove and weigh 10 g sample into a sterile
mortar containing 2 mL sterile mineral oil and 10 mL sterile Tween 20. Disperse with a ster-
ile spatula to form a paste. Add 78 mL sterile MLB to make a 10-1 dilution.
5.2.4 Cream and oil-based products. Aseptically remove and weigh 1 g sample into 20 x 150
mm screw-cap tube containing 1 mL sterile Tween 80 plus five to seven 5-mm glass beads

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(or ten to fifteen 3-mm glass beads). Mix total contents with Vortex mixer. Adjust total vol-
ume to 10 mL with sterile MLB (8 mL) for the 10-1 dilution.
5.2.5 Aerosols of powders, soaps, liquids, and other materials. Decontaminate nozzle of spray
can as much as possible by swabbing with gauze pad moistened with 70% (v/v) aqueous
ethanol. Expel some product to flush out nozzle; then spray appropriate amount into tared
dilution bottle, e.g., 1 g of product into 9 mL sterile MLB. Thoroughly mix product and
broth, and reweigh. This will be a 10-1 dilution if exactly 1 g of sample was obtained.
5.2.6 Anhydrous materials. Treat as in 5.2.2 or 5.2.4, as appropriate.

Note : For water immiscible samples : Transfer the samples to a suitable container containing
a suitable quantity of solubilizing agent (e.g. Tween 80). Disperse the sample within
the solubilizing agent and add appropriate volume of diluents or neutralizer.

5.3 Neutralization of the antimicrobial properties of the Cosmetic product

5.3.1 Any cosmetic products with anti-microbial properties (or preservatives) must be appropri-
ately neutralized before conducting the test. Any residual preservative should be checked by
re-challenging with appropriate microorganisms. Information relative to suitable neutraliz-
ers is given in Annex A.
5.3.2 Validation of the efficiency of neutralizer.
5.3.2.1 The two strains, representative of both Gram negative and Gram positive mi-
croorganism (e.g. Staphylococcus aureus ATCC6538, Pseudomonas aerugi-
nosa ATCC 9027) are used to demonstrate the validity of the neutralizer.
5.3.2.2 For each strain, mix the neutralized sample with a dilution of microorganism
(1x108 CFU/mL) and plate on a Petri dish. After incubation, check the nature
of the colonies and compare the count with a control (without sample). The
count should be less than 50% of the control.

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5.4 Enumeration

SAMPLE 10 g

5.4.1 Aerobic plate count

MLB
10 -1
90 mL

5.4.2 Yeast and

Mold Count

Fig.1 Enumeration diagram for cosmetic microbes. The diagram shows example of performing
serial dilution up to 10-3 with duplicate plate counts for each dilution.

5.4.1 Aerobic plate count (APC).

5.4.1.1 Spread Plate Technique
5.4.1.1.1 Use spread plate technique to facilitate recognition of different colony
types and, if necessary, for differential count. Prepare and label duplicate
sets of petri dishes containing modified letheen agar (MLA) for samples
at appropriate dilutions (10-1 to 10-6). Add either 1 or 2 mL of the cos-
metic preparation (refer 5.2 above) to 9 or 18 mL, respectively, of MLB,
for 10-2 dilution.
5.4.1.1.2 Dilute samples decimally in MLB (NOTE: save dilutions for enrichment
step) to obtain the appropriate dilution series (10-1 to 10-6). Begin with
10-2 if all the 10-1 dilution is used up.
5.4.1.1.3 Thoroughly mix dilutions and pipet 0.1 mL of each dilution onto surface
of solid media in prelabeled petri dishes.
5.4.1.1.4 Spread inoculum over entire surface with bent glass rod that was first
sterilized by dipping in 95% ethanol and quickly flamed to remove the

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ethanol. Use new spreader for each dilution (at low dilutions) because
some product residue may carry over and adversely affect the flame-
sterilization procedure. For effective inoculum absorption, be sure agar
surface is dried (30 min at 35°C) when agar is freshly made.
5.4.1.1.5 Let medium absorb inoculum before inverting and incubating plates for
24-48 hours at 35 ± 2°C.
5.4.1.1.6 Count all colonies on plates containing 25-250 colonies, and record re-
sults per dilution counted. Calculate and report the result with reference
to Annex B.

5.4.1.2 Pour Plate Technique

5.4.1.2.1 Use pour plate technique so that low level of microorganisms can be
counted. Prepare and label duplicate sets of Petri dishes for samples with
appropriate dilutions (10-1 to 10-5).
5.4.1.2.2 Prepare serial dilution by transferring 1 mL of the first dilution into 9 mL
suitable diluent. Mix the suspension using vortex mixer to ensure ho-
mogenous distribution.
5.4.1.2.3 Repeat the serial dilution process till the appropriate dilution is obtained.
5.4.1.2.4 Transfer 1 mL suspension from the serial dilution into the sterile petri
dishes.
5.4.1.2.5 Add about 20 mL of melted MLA to the petri dishes. Cover the petri dish
and carefully swirl the petri dish so that the inoculated sample mix well
with the media.
5.4.1.2.6 When the agar is set, invert the petri dishes and incubate at 35 ± 2°C for
24-48 hours.
5.4.1.2.7 Count all colonies in plates containing 25-250 colonies, and record results
per dilution counted. Calculate and report the result with reference to
Annex B.

If no colonies are obtained on MLA, observe already prepared MLB dilutions while
enriching them at 35 ± 2°C for 7 days. Examine enrichments daily for growth. After
7 days of incubation, or when growth is suspected, subculture all enrichments onto
MLA plates. Incubate plates at 35 ± 2°C for 48 hours.
5.4.2 Fungi, yeast, and mold plate count.
5.4.2.1 Transfer 0.1 mL portions of dilution series above to appropriately labeled
duplicate plates of either malt extract agar (MEA) or potato dextrose agar
(PDA), both containing 40 ppm chlortetracycline.
5.4.2.2 Spread inoculum over surface of medium with sterile glass spreader rod. Af-
ter inoculum is absorbed by medium, invert plates.
5.4.2.3 Incubate at 25 ± 2°C, and observe daily for 7 days.

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5.4.2.4 Average the counts obtained on duplicate plates, multiply by 10 to allow for
the volume plated (eg. 0.1 mL), multiply by the dilution factor, and report as
yeast or mold count/g (mL) sample.
5.4.2.5 For fungal enrichments (optional), dilute prepared sample decimally in
Sabouraud's dextrose broth and incubate as described above for MLB dilu-
tions.
5.4.2.6 If growth occurs, streak on Sabouraud's dextrose agar, MEA, or PDA. The
latter two agars should both contain 40 ppm chlortetracycline.

REMARKS

Cosmetic products are not expected to be aseptic. However, they must be completely free of high-
virulence microbial pathogens, and the total number of aerobic microorganisms per gram must be
low. The microorganisms which may be specified as pathogenic differ from country to country
according to the national practices or regulations. Pathogens or opportunistic pathogens whose inci-
dence would be of particular concern, especially in eye-area cosmetic products, include S. aureus,
Streptococcus pyogenes, P. aeruginosa and other species, and Klebsiella pneumoniae. Some mi-
crobes normally regarded as nonpathogenic may be opportunistically pathogenic.

6 REFERENCES

6.1 Anonymous. 2004. Microbial Limit Tests. In: United States Pharmacopeia, 27th Revision,
U.S. Pharmacopeial Convention, Rockville, MD. pp. 2152-2157.
6.2 Hitchins, A.D., T.T. Tran, and J.E. McCarron. 2001. Chapter 23 Microbiological Methods
for Cosmetics. In: U.S. FDA Bacteriological Analytical Manual Online.
6.3 Singer, S. 1987.The use of preservative neutralizers in diluents and plating media, Cosmetics
and Toiletries, 102, 55.
6.4 Compendium of methods for the microbiological examination of foods.4th ed. 2001. pp. 56-
57.

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Harmonised method:

• Issued by the microbial analysis group at the harmonization workshop in Kuala-

Lumpur, on September 13th to 17th, 2004

• Approved by the harmonization workshop delegates workshop in Kuala-Lumpur, on

September 13th to 17th, 2004,

• Modified after the Bangkok training, Nov 29th to Dec 3rd, 2004

• Modified (format only) in Brunei workshop Aug 30th to 31 st, 2005

• Modified and approved after the final review in Singapore, Nov 30th to Dec 2nd, 2005

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ANNEX A
(Informative)

Table A1. Neutralizers of antimicrobial activity of preservatives (ref. 5.3)

Chemical compounds able to neutalize
Preservative preservative’s antimicrobial activity
Examples of suitable neutralizers

Phenolic compounds : Lecithin, Polysorbate 80, 30 g/l + lecithin, 3 g/l.

Parabens, Polysorbate 80, Ethylene oxide condensate of fatty alcohol, 7
phenoxyethanol, g/l + lecithin, 20 g/l + polysorbate 80, 4 g/l.
Ethylene oxide condensate of fatty
phenylethanol,
alcohol D/E Neutralizing Broth
etc..Anilides
Non-ionic surfactants

Quaternary ammonium Lecithin, Saponine, Polysorbate Polysorbate 80, 30 g/l + sodium dodecyl
compounds, 80, sulphate, 4 g/l + lecithin, 3 g/l.
Sodium dodecyl sulphate,
Cationic surfactants Polysorbate 80, 30 g/l + saponin, 30 g/l +
Ethylene oxide condensate of fatty
lecithin, 3 g/l.
alcohol
D/E Neutralizing Broth

Aldehydes Glycine, Histidine Lecithin, 3 g/l + polysorbate 80, 30 g/l + L-

Formaldehyde-release Histidine, 1 g/l.
agents
Polysorbate 80, 30 g/l + saponin, 30 g/l + L-
Histidine, 1 g/l + L-cysteine, 1 g/l.
D/E Neutralizing Broth

Oxidizing compounds sodium thioglycollate Sodium thiosulphate, 5 g/l.

Isothiazolinones, Lecithin, Saponine, Amines, sul- Polysorbate 80, 30 g/l + saponin, 30 g/l +
Imidazoles fates,mercaptans, Sodium Bisulfite, lecithin, 3 g/l.
sodium thioglycollate

Lecithin, Saponine, Polysorbate Polysorbate 80, 30 g/l + saponin, 30 g/l +

Biguanides lecithin, 3 g/l.
80,

Metallic salts (Cu, Zn, Sodium bisulphate, cysteine Sodium thioglycollate, 0.5 g/l or 5 g/l.
Hg)
Sulfhydryl compounds, Thiogly- L-cysteine, 0.8 g/l or 1.5 g/l.
Organo-mercuric com-
collic acid
pounds D/E Neutralizing Broth

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ANNEX B

Guidelines for counting, calculating and reporting the amount of microorganisms (ref. 5.4)

To compute colony counts, multiply the total number of colonies (or the average number if replicate
plates of the same dilution are used) per plate by the reciprocal of the dilution used. Record the
dilution used and the number of colonies counted or estimated on each plate. To avoid giving false
ideas of precision and accuracy when computing colony counts, record only the first two left-hand
digits. Raise the second digit to the next highest number only when the third digit from the left is
5,6,7,8 or 9; use zeros for each successive digit to the right of the second digit (Table B1). Report
counts (or estimates thereof) as CFU per g or mL, as applicable.

When counts on duplicate plates or consecutive dilutions are averaged, round off counts to two
significant figures only at the time of conversion to the CFU per g (Table B1, Sample No. 1117).

The appropriate number of colonies to count on a plate is a function of colony size, plate size, and
size of differential properties produced on the medium. Typically, 25 to 250 colonies per plate yield
reliable results. Use this as a guide unless an alternate range is indicated for specific methods. The
following guidelines or “rules” should be used for selecting plates and calculating the CFU per g or
mL, as applicable:

1. One plate with 25 to 250 colonies. Select a plate with 25 to 250 colonies unless excluded by
spreaders or lab accidents (see Item 5). Count all colonies, including those of pinpoint size
and record the dilution used and the total number of colonies counted (Table B1, Sample
Nos. 1001, 1004, 1011 and 1012)

2. Duplicate plates. Count plates with 25 to 250 colonies and average the counts to obtain the
colony count (Table B1, Sample No. 1112). If only one plate of a duplicate pair yields 25 to
250 colonies, count both plates unless excluded by spreader and average the counts (Table
B1, Sample Nos. 1113 and 1114). When counting duplicate plates from consecutive decimal
dilutions, compute the count per g for each dilution and proceed as in Item 3 (Table B1,
Sample Nos. 1111, 1115, 1116 and 1117)

3. Consecutive dilutions with 25 to 250 colonies. If plates from two consecutive decimal dilu-
tions yield 25 to 250 colonies each, compute the count per g for each dilution and report the
arithmetic average as the CFU per g, unless the higher compute count is more than twice the
lower one. In that case, report the lower computed count as the CFU per g (Table B1, Sam-
ple Nos.1002, 1003, 1111, 1115, 1116 and 1117).

4. No plate with 25 to 250 colonies. If there is no plate with 25 to 250 colonies and one or
more plates have more than 250 colonies, select plate(s) having nearest to 250 colonies and
count as in Item g, for crowed plates. Report count as the estimated (est.) CFU per g (Table
B1, Sample Nos. 1009 and 1118)

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5. All plates have fewer than 25 colonies. If plates from all dilutions yield fewer than 25 colo-
nies, record the actual number of colonies on the lowest dilution (unless excluded by
spreaders) and report count as est.CFU per g (Table B1, Sample Nos. 1007 and 1119)

Table B1 Examples for computing colony count per gram or milliliter

Colonies counted
Sample no. Dilution a Colony countb
Count ratio (CFU/g or mL) Rule
1:100 1:1000
Common application, one plate from each of two dilutions
1001 234 23  23,000 1
1002 243 34 1.4 29,000 3
1003 140 32 2.3 14,000 3
1004 Sprd 31  31,000 1, 8
1005 0 0  <100 est. 6
1006 TNTC 7150  >5,600,000 est. 7
1007 18 2  1,800 est. 5
1008 Spr Spr  Spr 8
1009 325 20  33,000 est. 4, 7
1010 27 215  LA 8
1011 305 42  42,000 1
1012 243 LA  24,000 1, 8
1013 TNTC 840  840,000 est. 7

Procedure where two plates per dilution are poured

1111 228 28 1.2 25,000 2, 3
240 26
1112 175 16  19,000 2
208 17
1113 239 16  28,000 2
328 19
1114 275 24  30,000 2
280 35
1115 138 42 2.4 15,000 2, 3
162 30
1116 228 28 1.1 24,000 2, 3
240 23
1117 224 28 1.4 24,000 2, 3
180 Spr
1118 287 23  28,000 est. 4, 7
263 19
1119 18 2  1,700 est. 5
16 0
1120 0 0  <100 est. 6
0 0

a
Count ratio is the ratio of the greater to the lesser plate count, as applied to plates from consecutive dilutions having
between 25 to 250 colonies.
b
All count should bemade in accordance with rules listed or given in the text.
c
Underlined figures used to calculate count.
d
spr = Spreader and adjoining area of repressed growth covering more than one-half of the plate.

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e
LA = Laboratory accident. .

6. Plates with no colonies. If plates from all dilutions have no colonies and inhibitory sub-
stances have not been detected, report the estimated count as less than (<) one times the cor-
responding lowest dilution (Table B1, Sample Nos. 1005 and 1120)

7. Crowed plates (more than 250 colonies). If the number of colonies per plate exceeds 250,
count colonies in portion of the plate that are representative of colony distribution to esti-
mate the aerobic colony count. If there are fewer than 10 colonies per cm2 count the colonies
in 12 cm2, selecting six consecutive squares horizontal across the plate and six consecutive
squares

8. Spreaders. There are three distinct types of spreaders. The first type is a chain of colonies,
not too distinctly separated, that appears to be caused by disintegration of a bacteria clump
when the inoculum is dispersed in or on the plating medium. If one or more chains appear
to originate from separate sources, count each as one colony. Do not count each individual
colony in such chain(s) as separate colonies.

The second type of spreading colony develops in a film of water between the agar and the plate.
The third type forms in a film of water at the edge or over the surface of the agar. These two types
develop mainly because of moisture accumulation at the point from which the spreader originates,
and these spreaders may repress the growth of individual colonies. When dilution water is uni-
formly distributed throughout the medium, bacteria rarely develop into spreading colonies. Steps to
eliminate spreaders of this type should be taken if 5% of a laboratory’s plates have spreaders cover-
ing 25% of the plate.

If spreaders occur on the plate(s) selected, count colonies on representative portions thereof only
when colonies are well distributed in spreader free areas and the area covered by spreader(s), in-
cluding the total repressed growth area if any, does not exceed 50% of the plate area. Calculate the
estimated count by multiplying the average count per cm2 by the area of the plate. Where the re-
pressed growth area alone exceeds 25% of the total area, report as “spreaders”(spr) or “laboratory
accident” (LA) (Table B1, Sample Nos. 1008 and 1010)

Inhibitory substances in a sample may be responsible for the lack of colony formation. The analyst
may suspect the presence of inhibitory substances in the sample under examination when plates
show no growth or show proportionately less growth in lower dilutions. Such developments can-
not, however, always be interpreted as evidence of inhibition, and unless inhibition is demonstrated,
should be reported as LA.

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ANNEX C

Media and Reagents, (ref 3.1)

Letheen Agar (Modified)

Letheen agar (Difco or BBL) 32 g

Trypticase peptone 5 g
Thiotone peptone 10 g
Yeast extract 2 g
NaCl 5 g
Sodium bisulfite 0.1 g
Agar 5 g
Distilled water 1 liter
Heat with agitation to dissolve agar. Autoclave 15 min at 121°C. Aseptically dispense 20 mL into
15 x 100 mm petri dishes. Final pH, 7.2 ± 0.2.

Letheen Broth (Modified)

Letheen broth 25.7 g

Trypticase peptone 5 g
Thiotone peptone 10 g
Yeast extract 2 g
Sodium bisulfite 0.1 g
Distilled water 1 liter
Dispense 90 mL into screw-cap bottle. Autoclave 15 min at 121°C. Final pH, 7.2 ± 0.2.

Malt Extract Agar

(Cosmetics-General Microbiology)

Malt extract 30 g
Agar 20 g
Distilled water 1 liter

Boil to dissolve ingredients. Avoid overheating, which causes softening of agar and darkening of
medium color. Autoclave 15 min at 121°C. Dispense 20-25 mL into sterile 15 x 100 mm petri
dishes. Final pH, 5.5 ± 0.2.
For cosmetics use : Cool medium to 47-50°C after autoclaving. Dispense 4 mL stock filter-
sterilized chlortetracycline HCl solution (1 g/100 mL) per liter of medium to yield final concentra-
tion of 40 ppm chlorotetracycline HCl. Mix thoroughly and dispense 20 mL portions into 15 x 100
mm petri dishes.

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Potato Dextrose Agar

Potato infusion 200 g

Dextrose 20 g
Agar 20.0 g
Distilled water 1 liter
To prepare potato infusion, boil 200 g sliced, unpeeled potatoes in 1 liter distilled water for 30 min.
Filter through cheesecloth, saving effluent, which is potato infusion (or use commercial dehydrated
form). Mix in other ingredients and boil to dissolve. Autoclave 15 min at 121°C. Dispense 20-25
mL portions into sterile 15 x 100 mm petri dishes. Final pH, 5.6 ± 0.2.
Medium should not be re-melted more than once. Medium powder is available commercially but
may require supplementing with extra agar to a final concentration of 20 g/liter. To BBL or Difco
dehydrated medium, add 5 g of agar.
For cosmetics, cool medium to 47-50°C after autoclaving. Add 40 ppm (final concentration) chlor-
tetracycline. Mix thoroughly and dispense 20 mL portions into 15 x 100 mm petri dishes. Dispense
4 mL of stock filter-sterilized chlortetracycline HCl (1 g/100 mL) per liter of medium.

Sabouraud's Dextrose Broth and Agar

Polypeptone or neopeptone 10 g
Dextrose 40 g
Distilled water 1 liter
Dissolve completely and dispense 40 mL portions into screw-cap bottles. Final pH, 5.8. Autoclave
15 min at 118-121°C. Do not exceed 121°C.
For Sabouraud's dextrose agar, prepare broth as above and add 15-20 g agar, depending on gel
strength desired. Final pH, 5.6 ± 0.2. Dispense into tubes for slants and bottles or flasks for pour-
ing plates. Autoclave 15 min at 118-121 °C.

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